摘要
女贞子为木犀科女贞Ligustrum Lucidum Ait.的干燥成熟果实,是我国传统的扶正固本中药,味甘,苦,性凉,有养阴气,平肝火,壮筋骨,滋补肝肾等功效,应用十分广泛。临床主要用于治疗阴虚内热、眩晕耳鸣、腰膝酸软、须发早白、目暗不明等,并为相当多的中药方剂中的重要药味。现代药理研究表明,女贞子具有抑菌,缓下,保肝,升白,免疫调节等多方面的作用。
长期以来,一直认为女贞子的主要有效成分是以齐墩果酸为代表的五环三萜类化合物,并对其进行了较为深入的研究。但20世纪90年代后的国内外研究表明,女贞子中除低极性的齐墩果酸外,还含有较高含量的环烯醚萜苷类,以及红景天苷等多种水溶性成分。环烯醚萜苷具有保肝利胆,抗肝纤维化,抗菌消炎,抗肿瘤,降血脂,降血糖,降低血液粘度,增强机体免疫调节等多种药理活性,已日益引起广泛重视。而红景天苷是“高原人参”红景天的主要活性成分,女贞子环烯醚萜苷由多种化合物组成,两者化学结构具有共同特点,即分子中都含有对羟基苯乙基-β-D葡萄糖苷(即红景天苷)结构。因此,提取女贞子中环烯醚萜总苷作为中药有效部位研究,对于充分利用女贞子这一中药资源、和新药与新制剂的开发具有重要意义和广泛的前景。目前尚未见这方面的研究报道。
本研究建立了女贞子中红景天苷的含量测定方法,采用反相高效液相色谱法,建立的色谱条件为:采用Hypersil-C_(18)分析色谱柱(5μm,4.6mm×150mm),流动相为甲醇-水(20:80),流速为0.4mL/min,检测波长为220nm,进样量为20μL,灵敏度为0.010AUFS。本方法重复性好,精密度高,简便、快捷、准确,适用于红景天苷的含量测定。
本研究建立并优化了女贞子中红景天苷的分离纯化工艺。采用乙醇冷浸提取、大孔吸附树脂分离纯化的方法。提取物中红景天苷的含量可达到7.7%。目前只有报道从“高山红景天”中提取红景天苷,但“高山红景天”价格昂贵,资源稀少。本研究从资源丰富的女贞子中提取得到较高含量的红景天苷提取物,为工业化生产和女贞子资源的综合利用提供了科学依据。
本研究采用L_9(3~4)的正交实验因素水平表以提取时间、提取溶液体积、洗脱液中乙醇浓度作为考察因素。以薄层层析和薄层扫描法监测考察结果,以薄层层
The source of the crude drug, Frutus Ligustr Lucidi(Chinese name, Nuzhenzi ) are the fruits of Ligustrum Lucidum Ait which is well known and frequently used in clinical practice.In the theory of Chinese traditional medicine, it has the effects of supporting the healthy energy, nourishing the liver and kidneys and can be applied to replenish the liver and kidney, improve eyesight and promote the growth of black hair. It is also used as an important component in some traditional Chinese patent medicines. Pharmacological tests show that it has the activities to improve immune function and delay the ageing process.Ever since a long time ago, it is considered that oleanolnic acid and the other compound is the active substance in the fruits of Ligustrum Lucidum Ait. However, after 20 century the world research indicate that besides the low-polar constituent oleanolnic acid, the fruits of Ligustrum Lucidum Ait is also contains water-solubility components, such as high-contents iridoid glcosides, saldroside and so on . Iridoid glcosides has many biological functions like protect liver and cholagogue, anti-hepatic fibrosis, antibiosis and dephologisticate, subdue blood-fat, cut down blood-sugar, degrade viscosity, reinforce immunological regulation, etc. Salidroside is the main active component of Rhadolia rasea L., iridoid glycosides in the fruits of Ligustrum Lucidum Ait. is consists of many compounds which have the same chemical constitution- p-hydroxyphenethyl- β -D-glucoside. So, consider iridoid glcosides in the fruits of Ligustrum Lucidum Ait as traditional Chinese drug effective elments is very important for make best of Ligustrum Lucidum Ait, have significance meaning for the development of new drug and new preparation.To establish a RP-HPLC method for determining the content of saldroside in fruits of Ligustrum Lucidum Ait. The RP-HPLC separation was accomplish on Hypersil-C_(18) column(5μm,4.6mm×150mm) with mobile phase of methanol water (20:80) by flw rate of 0.4 mL/min.The detection wavelength was 220nm. This method is proved to be simple, rapid, accurate and suitable for the qualty control of salidroside in Ligustrum Lucidum Ait.
To optimize the purified process for salidroside in Ligustrum Lucidum Ait. Compounds were extracted with ethanol and purified by macroreticular resin. The content of extration can reach 7.7%. Now salidroside was only extract from Rhadolia rasea L. which is rare and expensive. Ligustrum Lucidum Ait which is abundance in our country is adopted to extract salidroside with high contents. It provides a scientific basis for the commercial production and utilization of Ligustrum Lucidum Ait.The orthogonal design (with L9 (34) orthogonal table) was used to optimize the extraction process of total glycosides from Ligustrum Lucidum Ait, three factors in extraction were optimized, i.e., extraction times, volume of extraction system and concentration of ethanol of elutant. The scan response of the spot of the salidroside and high polar components in TLC was used as a index, the methods of extraction including reflux > cold-soaked ^ water extract and alcohol deposited determined by TLC and Scan-TLC. The result shows that by cold-soaked extraction way the optimum extraction condition obtained is as follows: alcohol concentration of 70 %, cold-soaked time of 48h and extraction times of 3 times. And the content of total iridoid glycosides in Ligustrum Lucidum Ait is the highest and the purification is the easiest.For the properties of macroreticular resin, the influencing factors such as alcohol concentration of eluant, temperature, equilibrium time, and concentration of eluant, quantity of swilled alcohol, the ratio between extraction and macroporous resin, adsorption time and the flow rate on desorption were studied and the optimal process route and conditions were suggested in this paper: Ligustrum Lucidum Ait was absorbed for 30 minutes and washed with 500mL distilled water, then eluted with 200mL of 10%-, 30%^ 50% ethanol, and finally collected the eluant of 50% ethanol to evaporate to dryness with aqueous bath. Put the collection in polyamide column, eluted with lOOmL distilled water and get the dried product was the extraction of iridoid glycosides of Ligustrum Lucidum Ait.According to the methods of extraction, we established new process chart for the extraction > separation and purification of iridoid glycosides of Ligustrum Lucidum Ait. The average content of total iridoid glycosides in the extraction was 65.7% and the average extraction rate was 10.76mg/g.
We established Alkaline hydrolysis -RP-HPLC for measuring the total iridoid glycosides in the extraction of Ligustrum Lucidum Ait. That is to say after alkaline hydrolysis, determine the quantity of salidroside with RP-HPLC and calculate the content of iridoid glycosides. The concrete procedure of this method is that dissolve about 20mg extraction with 2mL 5% NaoH, then heat in aqueous bath for lh, cool down, and use diluted hydrochloric acid limestone to neutralize the pH value to 7.In this paper, we summarized the characters such as identification, determination and quality control standards to the extraction of iridoid glycosides of Ligustrum Lucidum Ait.lt can be the guarantee of the stability and security of the finished product and also be a base for the further qualitative or quantitative analytical research.
引文
[1] 江立虹.女贞子开发研究现状及前景.江西林业科技,2004(4):46-48
[2] 张萍,冯琴琴.女贞子的药理与临床应用研究概况.中国药业,2002,11(4):78-80
[3] 蔡立明.女贞子药理作用及其临床应用.时珍国医国药,2000,11(5):474-475
[4] 李葆华.女贞子的研究状况.中华医学研究与实践,2004,2(7):49-51
[5] 黄婉,杨耀芳.女贞子及其有效成分的药理及临床研究进展.现代中西医结合杂志,2003,12(7):772-774
[6] 石力夫,蔡溱,曹颖瑛,王鹏.中药女贞子的化学成分及其药理作用.药学实践杂志,1997,15(4):197-200
[7] 石力夫,曹颖瑛,陈海生,董建萍.中药女贞子水溶性成分二种新裂环环烯醚萜甙的分离和鉴定.药学学报,1997,32(6):442-446
[8] 陈千良,孙文基.裂环烯醚萜类化合物研究进展.国外医药·植物药分册,2003,18(2):58-63.
[9] 董娟娥,张靖.植物中环烯醚萜类化合物研究进展.西北林学院学报,2004,19(3):131-135 Journal of Northwest Forestry University
[10] 刘净,梁净钰,谢韬.环烯醚萜类化合物近年研究进展.海峡药学,2004,16(1):14-19
[11] 乔卫,张彦文,吴寿金.天然环烯醚萜类化合物的生物活性.国外医药·植物药分册,2001,16(2):65-67
[12] 王树海,王文健,汪雪蜂,陈伟华.红景天苷对3T3-L1脂肪细胞糖代谢及细胞分化的影响.中西医结合学报,2004,2(3):193-195
[13] 张文生,朱陵群,牛福玲,邓瑞春,马传香.红景天苷对缺氧/缺糖损伤神经细胞的保护作用.中国中药杂志,2004,29(5):459-462
[14] 王晓东,刘永刚,苏薇薇.红景天苷对小鼠实验性肝损伤的保护作用.中药材,2004,27(3):198-199
[15] 胡敏,于金德.红景天苷对心血管系统作用的研究.国外医学·心血管疾病分册,2003,30(5):298-301
[16] 钟显飞,蒋明德,马洪德,曾维政.红景天苷对乙醛刺激的大鼠肝星状细胞调亡的影响.中药新药与临床药理,2004,15(3):161-164
[17] 王晓东,刘永忠,刘永刚.红景天苷体外抗肝纤维化的实验研究.时珍国医国药,2004,15(3):138-139
[18] 曲智威.红景天的研究进展.中国实用中西医杂志,2005,18(7):1063-1065
[19] 赵永唤,刘成海,武廷华.红景天的研究与进展.中国林副特产,1998(3):44-45)
[20] 邰韧辉.高纯度红景天苷的制备工艺研究.现代中药研究与实践,2004,18(4):58-59.
[21] 李静,鱼红闪,张春枝,等.制备红景天甙标准品的新方法.大连轻工业学院学报,2002,21(3):189-192.
[22] 许建峰,应佩青,苏志国.高山红景天资源应用与开发研究进展.中草药,1998,29(3):202-205
[23] 王强,阮晓,方兰,颜启传.资源植物红景天研究综述.新疆农业大学学报,2002,25(4):57-62
[24] 李伟,黄勤妮.红景天属植物的研究及应用.首都师范大学学报(自然科学版),2003,24(1):55-59
[25] 孙福岭,肇责超.高山红景天及其栽培技术.内蒙古农业科技2000,(5):45
[26] 李葆华.女贞子的研究状况.中华医学研究与实践,2004,2(7):49-51
[27] 赵继霄,杨谨,董根玲,王乃婕.女贞子研究进展.中华医学写作杂志,2004,11(18):1599-1601
[28] 胡馨,张国明,童月建.红景天片剂薄层鉴别及红景天甙的含量测定.中成药,2000,22(11):808-810
[29] 卢水昌,林鹏程,鲍寿涛.大花红景天中红景天甙的提取及含量测定.青海师范大学学报(自然科学版),2005,(1):34-37
[30] 刘传学,肖德华,刘德文.HPLC法测定高山红景天中红景天苷的含量.中国林副特产,2005,(4):9
[31] 毛红霞,黎源倩,曾红燕,等.保健食品中红景天甙的提取和测定方法研究.中国卫生检验杂志,2004,14(1):10-12
[32] 刘永刚,金向群,叶谨,等.大孔树脂纯化赤芍总苷的研究.中药材,2005,28(3):195-196
[33] 才谦,刘涛,付玉芹,等.大孔吸附树脂法富集龙胆中裂环烯醚萜苷类成分的实 验研究.2003,25(4):271-272
[34] 皮文霞,蔡宝昌,潘扬,等.大孔树脂与活性炭富集山茱萸总苷的实验研究.中国中药杂志,2002,27(8):583-584
[35] 汪程远,张浩,孟莉,等.大孔吸附树脂分离纯化生地黄中苷类与糖类.中药材,2003,26(3):202-204
[36] 马双成,邓少伟.赤芍总苷的生产工艺条件研究.中草药,1998,29(10):664-667
[37] 刘臣,张钧寿.大孔吸附树脂分离纯化刺五加苷D的研究.中成药,2004,26(9):701-704
[38] 杨荣平,王宾豪,方艾权,等.大孔树脂分离葛根总黄酮工艺优化.中成药,2004,26(10):754-757
[39] 石召华,熊富良,李崇明,等.大孔吸附树脂分离纯化三七总皂苷工艺研究.中成药,2004,26(1):10-12
[40] 尹蓉莉,江靖,张淑儒,等.大孔吸附树脂纯化白芍总苷的工艺研究.时珍国医国药,2005,16(7):610-612